PHYSIOLOGIA PLANTARUM 109: 359–371. 2000
Copyright © Physiologia Plantarum 2000
ISSN 0031-9317
Printed in Ireland —all rights reser6ed
Minireview
Circadian rhythms in plants: a millennial view
C. Robertson McClung
Department of Biological Sciences, Dartmouth College, Hano6er, NH 03755 -3576, USA
*E-mail: [email protected]
Received 6 December 1999; revised 10 January 2000
Circadian rhythms are endogenous rhythms with periods of
approximately 24 h. These rhythms are widespread both
within any given organism and among diverse taxa. As genetic
and molecular biological studies, primarily in a subset of
model organisms, have begun to identify the components of
circadian systems, there is optimism that we will soon achieve
a detailed molecular understanding of circadian timing mechanisms. Although plants have provided many examples of
rhythmic outputs, and our understanding of photoreceptors of
circadian input pathways is well-advanced, plants have lagged
behind other groups of organisms in the identification of
components of the central circadian oscillator. However, there
are now a number of promising candidates for components of
plant circadian clocks, and it seems probable that we will soon
know the details of a plant central oscillator. Moreover, there
is also accumulating evidence that plants and other organisms
house multiple circadian clocks, both in different tissues and,
quite probably, within individual cells. This provides an unanticipated level of complexity with the potential for interaction
among these multiple oscillators.
Introduction
Most organisms exhibit temporal organization of their activities with respect to the environmental oscillation of day and
night, and thus express diurnal rhythms. When deprived of
environmental time cues, such as light-dark or temperature
cycles, many of these rhythms persist, indicating that organisms have the endogenous capacity to measure time and to
use this time information to temporally regulate their biology. Circadian rhythms are a subset of endogenous rhythms
with periods of approximately 24 h (Pittendrigh 1981b,
Edmunds 1988, Johnson et al. 1998). The deviation of the
free-running period from exactly 24 h has been termed the
‘‘strongest single piece of evidence that the overt rhythm is
under the control of an endogenous timing mechanism’’
(Feldman 1982) because this finding makes it clear that
circadian rhythms are truly intrinsic, and do not result from
the organism deriving time cues from some subtle geophysical signal originating with the 24 h period of the rotation of
the earth. The period of a circadian rhythm remains relatively constant over the range of physiologically relevant
temperatures (exhibits temperature compensation), which
makes intuitive sense; a reliable clock should not run faster
or slower in response to the vagaries of the weather, or in
response to the passage of the sun behind a cloud. However,
both abrupt temperature changes or temperature cycles as
well as pulses or cycles of light act as potent stimuli that can
shift the phase of the clock. The primary effect of the
environmental time cues associated with the diurnal cycle is
to entrain the endogenous timing system to a period of 24 h
(Pittendrigh 1981a), which precisely corresponds to the environmental period resulting from the rotation of the earth on
its axis.
The ubiquity of circadian rhythmicity across a broad
taxonomic spectrum has prompted the speculation that
adaptive fitness is enhanced by the synchronization of an
organism’s internal clock with the diurnal cycle imposed by
its environment. The scientific testing of this hypothesis
largely has been avoided and one has been forced to rely on
the adage ‘‘the early bird gets the worm’’ in any effort to
justify the adaptive fitness of aspects of circadian biology,
such as dawn anticipation. However, the study of the circadian biology of cyanobacteria (Johnson et al. 1998, Johnson
and Golden 1999) has opened the door to rigorous scientific
Abbre6iations – CAM, crassulacean acid metabolism; L:D, light:dark; LUC, luciferase; QTL, Quantitative Trait Locus.
Physiol. Plant. 109, 2000
359
testing of the adaptive significance of circadian rhythmicity
in studies that recapitulate evolutionary events in the laboratory. Mutants of Synechococcus sp. strain PCC7942, with
alterations in period length, have been identified on the basis
of the rhythmic expression of a photosynthetic gene fused to
bacterial luciferase (luxAB from Vibrio har6eyi ), a reporter
that allows non-invasive measurement of gene expression in
real time (Kondo et al. 1994). Strains with wild type (25 h),
short (23 h) or long (30 h) period grow at essentially the
same rate in pure culture in either continuous light or in
light-dark cycles. However, when these strains are mixed
and competed against each other in either 22 h (light:dark
[L:D] 11:11), 24 h (L:D 12:12), or 30 h (L:D 15:15) cycles, in
each case the strain whose period most closely matches that
of the environmental L:D cycle eliminates the competitor
(Johnson and Golden 1999). The rapidity with which the
successful strain eliminates its competitor implies that the
selective coefficient is unexpectedly high. The mechanism of
this fitness enhancement remains poorly understood (Johnson and Golden 1999). Nonetheless, it is gratifying to the
circadian biologist that the circadian system is significant in
Darwinian terms.
It is possible, at least formally, to divide the circadian
system into 3 conceptual parts: an input pathway or set of
pathways that provide temporal information from the environment to the clock, the central oscillator (‘clock’) mechanism itself, and an output pathway or set of pathways
through which the temporal information provided by the
clock is used to generate overt rhythms in various processes
(Fig. 1). In practice, the assignment of a component gene or
gene product to a role in input, oscillator or output is often
complicated as input pathways can themselves be regulated
by the clock, and outputs often feedback to regulate inputs.
Nonetheless, this conceptual view of the circadian system
provides a useful framework within which to organize the
consideration of circadian timekeeping. In this review, I will
first address the central oscillator mechanism as it has been
described in Drosophila in order to familiarize the reader
with a reasonably well-understood molecular oscillator. Second, I will discuss plant clock outputs, as this will introduce
the overt rhythms that have been studied. Then I will
examine input pathways, and finally I will return to a
consideration of the plant oscillator mechanism.
The oscillator: a negative feedback loop? The
paradigm in cyanobacteria, fungi, flies and mammals
For many years the Holy Grail of clock research has been
the identification of components of the central oscillator. A
combination of mutational analyses, chiefly in Drosophila,
Neurospora, and Synechococcus, together with molecular
analyses both in these systems and mammals, has yielded a
model of the central oscillator as a negative feedback loop
in which rhythmic transcription of key clock genes is inhibited by the nuclear accumulation of the protein products of
these genes. Please note that in the following description (see
Fig. 2) of the Drosophila version of this negative feedback
cycle, I will only cite those primary references that have
been published since the excellent recent reviews by Young
(1998) and Dunlap (1999). During the subjective day phase,
a heterodimer of the transcription factors Drosophila
CLOCK (dCLK) and CYCLE (CYC) binds to conserved
E-boxes (Hao et al. 1999) to transcriptionally activate PERIOD (PER) and TIMELESS (TIM). The dCLK/CYC
heterodimer also negatively regulates dCLK transcription,
although this may be indirect (Glossop et al. 1999). PER is
unstable in the cytoplasm, but becomes stabilized by heterodimerization with TIM. Upon reaching a threshold concentration, the PER/TIM heterodimer translocates into the
nucleus. This process introduces a time delay that is critical
to the generation of a 24-h periodicity. The PER/TIM
heterodimer is phosphorylated by DOUBLETIME (DBT), a
casein kinase type Io. The phosphorylated PER/TIM heterodimer negatively regulates the dCLK/CYC heterodimer
through formation of a larger complex. This both blocks the
positive regulation of PER and TIM transcription and
relieves the negative regulation of dCLK transcription (Bae
et al. 1998, Glossop et al. 1999). At dawn, the Drosophila
photoreceptor, CRYPTOCHROME (dCRY), undergoes a
photochemical change that allows it to interact with TIM,
Fig. 1. Conceptual model of a simple
circadian system consisting of a set of
input (entrainment) pathways, a central
oscillator, and a set of output pathways.
Entraining stimuli include light, mediated
through phytochromes (PHY) and
cryptochromes (CRY), and temperature.
Although the input pathways are drawn
as discrete linear pathways, there are
multiple phytochromes and
cryptochromes as well as interaction
among them and their downstream
signaling pathways. The central oscillator
is illustrated as a loop, including positive
and negative components that yields a
self-sustaining oscillation with a period of
approximately 24 h. Multiple output
pathways are drawn as each regulating an
overt rhythm with a distinct phase. The
number of output pathways and the
degree of interaction among them is not
known, although some cross-talk among
output pathways is possible.
360
Physiol. Plant. 109, 2000
regulators has been conserved and offers a likely paradigm
for consideration in our pursuit of the plant central
oscillator.
Output rhythms
Growth rhythms
Fig. 2. A model of the Drosophila central oscillator. The CYCLE
(CYC)/dCLOCK (dCLK) heterodimer binds to E-box promoter
elements and is a positive regulator of PERIOD (PER) and TIMELESS (TIM) transcription, but is a negative regulator of dCLK
transcription, indicated by the X through the transcriptional arrow.
PER and TIM protein accumulate in the cytoplasm, although PER
is destabilized through DOUBLETIME (DBT)-mediated phosphorylation, indicated by the P-containing circles. Heterodimerization
of PER with TIM stabilizes PER and allows PER accumulation,
although the kinetics of heterodimerization and nuclear localization
introduce a lag in the accumulation of PER. The PER/TIM heterodimer complexes with the CYC/dCLK heterodimer, abrogating
positive regulation of PER and TIM transcription and relieving
negative regulation of dCLK transcription. Light activates CRYPTOCHROME (CRY), which sequesters TIM, which is ubiquitinated and degraded via the 26S proteasome. Removal of TIM from
the PER/TIM heterodimers allows renewed transcription through
the association of CYC/dCLK heterodimers with E-box containing
promoters.
inactivating the PER/TIM complex and relieving the negative regulation of dCLK/CYC transcription of PER and
TIM (Ceriani et al. 1999). TIM is degraded in the light by
an ubiquitin-proteasome mechanism (Naidoo et al. 1999).
This affords the view of the Drosophila oscillator as
comprised of two interlocked feedback loops in which two
heterodimers, PER/TIM and dCLK/CYC, are positive regulators of each other and negative regulators of themselves
(Glossop et al. 1999). Some key clock components (PER,
TIM, dCLK) are regulated by the clock at the level of
mRNA accumulation and protein accumulation or activity
whereas other key components (CRY, CYC, DBT) do not
oscillate. Furthermore, a number of the key components are
remarkably well-conserved between Drosophila and mammals (Dunlap 1999). In contrast, there is little obvious
conservation of these components with those identified to
date in the Neurospora and cyanobacterial clocks, although
Neurospora does employ the PAS domain, a protein-protein
interaction motif originally identified in PER and also found
in dCLK and CYC. Nonetheless, it seems that the basic
mechanism of a negative feedback loop inhibiting positive
Physiol. Plant. 109, 2000
Most studies of plant leaf movements have addressed the
pulvinar sleep movements in which cells in the extensor and
flexor regions of the pulvinus swell in antiphase (180° out of
phase), mediating a circadian oscillation in leaf position
(Satter et al. 1990, Engelmann and Johnsson 1998). Swelling
is driven by ion (chiefly potassium) fluxes (Kim et al. 1993).
These volume changes of flexor and extensor cells persist in
protoplasts in constant conditions, providing an excellent
system in which to study the roles of second messengers,
including calcium and phosphoinositides (Mayer et al.
1997).
It has recently been demonstrated that there are circadian
oscillations in cotyledon position in Arabidopsis that are
thought to arise from oscillations in elongation rates of
abaxial and adaxial cells of the petiole (Engelmann et al.
1992, Engelmann and Johnsson 1998). As these oscillations
are in elongation rates, I will consider these so-called ‘leaf
movement’ rhythms together with circadian rhythms in
hypocotyl and inflorescence stem elongation. Leaf movements of individual seedlings are easily monitored by video
imaging (see http://www.dartmouth.edu/rmcclung/leafmovement.html), allowing a high throughput assay to evaluate the effects of mutations on fundamental clock
properties, including period, phase, and temperature compensation. Recently, the rhythm in leaf movement was
exploited as the basis for a search for natural alleles that
contribute quantitatively (quantitative trait loci, or QTLs)
to the circadian period length in Arabidopsis (Swarup et al.
1999).
There is a circadian rhythm in the elongation rate of the
stem of Chenopodium rubrum (Lecharny and Wagner 1984)
that is correlated with a rhythm in oleic acid content
(Lecharny et al. 1990). More recently, it has been shown
that there is a rhythm in floral stem elongation in Arabidopsis (Jouve et al. 1998). This oscillation in stem growth rate is
correlated with an oscillation in the level of indole-3-acetic
acid (IAA) in rosette leaves, but not, curiously enough, in
the inflorescence stem itself. Decapitation of the inflorescence stem abolishes elongation; application of IAA, but not
the IAA-aspartate conjugate, to the surface of the decapitated stem restores both elongation and the oscillation in the
rate of elongation (Jouve et al. 1999). This argues that the
rhythm in elongation does not result from rhythmic synthesis of IAA in the shoot apex, but rather reflects a rhythm
either in polar transport of IAA or in the ability to elongate
in response to IAA. The IAA polar transport inhibitor
N-(1-naphthyl)phthalamic acid (NPA) blocks elongation
and evidence of a rhythm, indicating that polar transport of
auxin from the apex is necessary for elongation, but this
does not allow one to distinguish between either rhythmic
IAA transport or sensitivity as critical for the overt rhythm
in elongation rate.
361
It has long been known that light signaling leads to a
cessation of hypocotyl elongation and this phenotype has
been effectively exploited to identify components of the light
perception and signal transduction pathways through forward genetic (mutational) analysis. Mutations that reduce
photoperception and subsequent signaling yield an elongated hypocotyl phenotype; conversely, overexpression of
elements of the phototransduction pathways reduces
hypocotyl elongation relative to wild type. An elongation of
the hypocotyl also frequently accompanies a disruption of
circadian rhythmicity. For example, early flowering 3 (elf 3 )
mutants are conditionally arrhythmic in continuous light
and display an elongated hypocotyl (Hicks et al. 1996). It
was initially suspected that this reflected alterations in photoperception or transduction of the photic signal. However,
mutants overexpressing LATE ELONGATED HYPOCOTYL (LHY) (Schaffer et al. 1998) or CIRCADIAN
CLOCK ASSOCIATED 1 (CCA1 ) (Wang and Tobin 1998)
are arrhythmic and also display elongated hypocotyls, suggesting a more direct link between hypocotyl elongation and
the circadian system. Indeed, careful analysis of hypocotyl
elongation, using a video imaging system developed to
monitor leaf movements, demonstrated that the rate of
hypocotyl elongation upon germination is regulated by the
circadian clock with an episode of rapid elongation at
subjective dusk and a daily growth arrest at subjective dawn
(Dowson-Day and Millar 1999). In at least some mutants,
the hypocotyl elongation defect may result from a primary
dysfunction in the circadian system with a resulting failure
to impose a daily period of growth arrest (Dowson-Day and
Millar 1999).
Stomatal movements, gas exchange and CO2 assimilation
Circadian rhythms in stomatal aperture and in the responsiveness of the guard cells to environmental stimuli have
been described in a number of systems (Gorton 1990, Webb
1998). In beans, there is circadian control of the underlying
biochemical reactions of the Calvin cycle in addition to
control of stomatal aperture and gas exchange (Hennessey
and Field 1991). Although Arabidopsis exhibits a circadian
rhythm in the rate of CO2 fixation (C.R. McClung, unpub-
lished), it is not known if this rhythm includes circadian
regulation of the Calvin cycle reactions. Circadian regulation of sucrose metabolism has been demonstrated in
tomato (Jones and Ort 1997, Jones et al. 1998).
Rhythms in gene expression: transcriptional rhythms
Circadian control of transcription is widespread (Dunlap
1999). The most extreme example yet described is the
cyanobacterium Synechococcus, where circadian regulation
of transcription may be global (Johnson and Golden 1999,
Kondo and Ishiura 1999). It is important to note that the
peaks in mRNA abundance of different genes can occur at
distinct circadian phases. For example, mRNA abundance
of the CAT2 and CAT3 catalase genes of Arabidopsis peaks
at dawn and dusk, respectively (Zhong and McClung 1996).
The list of plant genes regulated by the circadian clock is
already extensive (Kreps and Kay 1997, Fejes and Nagy
1998). For example, many genes with photosynthetic roles
show circadian regulation of transcript abundance (Giuliano
et al. 1988, Martino-Catt and Ort 1992, Pilgrim and McClung 1993, Salvador et al. 1993, McClung 1997, Scandalios
et al. 1997, Nakahira et al. 1998, Piechulla 1999, McClung
et al. 2000). These include genes with roles in light harvesting, oxygen evolution, ATP synthesis, carbon assimilation
and photorespiration (Table 1). Although most genes exhibiting circadian oscillations are encoded in the nucleus,
there are also oscillations in abundance of chloroplast transcripts in wheat (Nakahira et al. 1998) and in Chlamydomonas (Salvador et al. 1993, Hwang et al. 1996). In
Chlamydomonas, these oscillations are correlated with and
may derive from a circadian oscillation in DNA supercoiling
in the plastid genome (Salvador et al. 1998). With the
imminent completion of the Arabidopsis genomic sequence it
seems highly probable that a microarray strategy (Kehoe et
al. 1999) will soon define the proportion of the Arabidopsis
genome that is under circadian control at the level of
oscillations in mRNA abundance.
Of course, oscillations in mRNA abundance can arise
from differential rates of transcription or from differential
mRNA stability. To date, studies in plants that have moved
beyond the simple description of a circadian oscillation in
Table 1. Photosynthetic genes exhibiting circadian oscillations in transcript abundance.
Role
Gene
Genome
Gene product
Species
Reference
psbD
LHCA
LHCB
Oxygen evolution OEE1
ATP synthesis
ATPA
ATPB
CO2 assimilation RBCS
RBCL
RCA
Nuclear
Nuclear
Nuclear
Nuclear
Chloroplast
Chloroplast
Nuclear
Chloroplast
Nuclear
Photosystem II (PSII) subunit
Chlorophyll a/b binding (PSI)
Chlorophyll a/b binding (PSII)
O2-evolving enzyme protein 1
ATP synthase
ATP synthase
Rubisco small subunit
Rubisco large subunit
Rubisco activase
Photorespiration
CAT
Nuclear
Catalase
SHM
Nuclear
Serine hydroxymethyltransferase
Wheat
Tomato
Many
Tomato
Chlamydomonas
Chlamydomonas
Arabidopsis
Chlamydomonas
Tomato
Arabidopsis
Maize
Arabidopsis
Arabidopsis
Nakahira et al. 1998
Kellmann et al. 1999
Piechulla 1999
Giuliano et al. 1988
Salvador et al. 1993
Salvador et al. 1993
Pilgrim and McClung 1993
Salvador et al. 1993
Martino-Catt and Ort 1992
Pilgrim and McClung 1993
Scandalios et al. 1997
McClung 1997
McClung et al. 2000
Light harvesting
362
Physiol. Plant. 109, 2000
mRNA abundance have focused on transcriptional control.
The first description of a circadian oscillation in mRNA
abundance of a plant gene was of a chlorophyll a/b binding
protein gene (CAB or LHCB) of pea (Kloppstech 1985).
Circadian oscillations in LHCB mRNA abundance has
proven a general phenomenon (Fejes and Nagy 1998,
Piechulla 1999); all 19 of the tested tomato LHCB genes
showed essentially identical circadian mRNA oscillations
(Kellmann et al. 1993). Both nuclear run-on experiments
and transcriptional promoter-reporter gene fusions have
established a transcriptional component to this regulation in
several plant species (Fejes and Nagy 1998, Piechulla 1999).
Minimal LHCB promoters sufficient to confer circadian
transcription have been identified in wheat (Nagy et al.
1988, Fejes et al. 1990), tomato (Piechulla et al. 1998) and
Arabidopsis (Millar and Kay 1991). These studies were aided
enormously by the development of firefly and beetle luciferases (LUC) as reporter genes. Typical reporter genes are
unsuitable for these studies because the stability of the
protein they encode (e.g. b-glucuronidase or chloramphenicol acetyltransferase) is too great to allow turnover within a
circadian cycle. Even though mRNA abundance oscillates in
response to rhythmic transcription, the reporter activity is
stable and accumulates over time, which obscures the underlying rhythm in transcription. Luciferase protein itself is
stable and western blot analysis indicates that it accumulates
over time. However, luciferase activity (light production) is
quite unstable. This renders light production dependent
upon de novo translation, thus allowing light production to
accurately track the circadian rhythm in LHCB transcription (Millar et al. 1992a,b). The measurement of luciferase
activity is non-destructive and allows quantitative resolution
of gene expression in both temporal and spatial terms in real
time in individual seedlings. This has proven a boon to both
the definition of cis-acting elements and to the identification
of mutants with defects in circadian timing.
In vivo analysis of progressively truncated LHCB1*1
(CAB2 ) promoter fragments fused to the luciferase gene
defined a 36-bp region sufficient to confer circadian transcription. In vitro analysis of DNA-binding by electrophoretic mobility shift assays and DNA footprinting
identified binding sites for multiple complexes in this short
fragment (Anderson et al. 1994, Anderson and Kay 1995,
Carré and Kay 1995). A single Myb domain transcriptional
activator encoded by the CCA1 gene that had been previously implicated in phytochrome regulation (Wang et al.
1997) shows circadian binding to an element (consensus
AAa/cAATCT) within the functionally defined region of the
LHCB1*1 promoter (Wang and Tobin 1998). CCA and the
closely related LHY (Schaffer et al. 1998) define a growing
family of single Myb domain proteins that figure prominently in the Arabidopsis circadian system, as will be discussed in greater detail below.
Minimal promoter fragments (278 and 119 bp) have also
been defined for two tomato LHCA genes (Kellmann et al.
1999). A minimal (330 bp) clock-responsive promoter has
been defined for the Arabidopsis RCA gene, whose expression oscillates in phase with the LHC genes (Liu et al. 1996).
Each of these promoter fragments includes a consensus
CCA1 binding site, although the functional importance of
Physiol. Plant. 109, 2000
CCA1 binding to the circadian transcription of LHCA or
RCA has not yet been experimentally demonstrated.
In addition to the LHCB and RCA promoters, which
confer morning-specific transcription, 3 dusk-specific promoters have also been defined. To confer circadian transcription with a dusk-specific phase, 490 bp of the
Arabidopsis GER3 promoter are sufficient (Staiger et al.
1999); 265 bp of the Arabidopsis GRP7 (CCR2 ) promoter
confer a robust high amplitude rhythm with dusk-specific
phase, and as few as 56 bp are sufficient to confer a low
amplitude (about twofold) oscillation (Staiger and Apel
1999). This smallest promoter fragment retains a CCA1
binding site, as does a minimal promoter (230 bp) of the
CAT3 catalase gene that is sufficient to confer dusk-specific
transcription (T.P. Michael and C.R. McClung, unpublished). The functional significance of the CCA1 sites in the
dusk-specific promoters has not been directly tested, but it is
intriguing that the same CCA1 binding site can be found in
promoters that are transcribed nearly 180° out of phase with
one another. The elucidation of the mechanism by which the
phase of transcription is determined is an interesting puzzle
that will undoubtedly be the focus of considerable experimental effort.
Rhythms in gene expression: post-transcriptional rhythms
One of the best-defined systems of post-transcriptional regulation is the activation state of phosphoenolpyruvate (PEP)
carboxylase. In crassulacean acid metabolism (CAM) plants,
PEP carboxylase activity is driven by a circadian rhythm in
the phosphorylation state of the enzyme that, in turn,
reflects the activity of a protein kinase (Nimmo 1998). PEP
carboxylase kinase itself is regulated at the level of translatable mRNA (Hartwell et al. 1996). Phosphorylation is increasingly prominent as a mechanism of circadian
post-translational regulation: recent studies demonstrate circadian regulation of sucrose phosphate synthase activity in
tomato by protein phosphatase activity (Jones and Ort
1997).
Post-transcriptional regulation can either obscure or contribute to oscillations in mRNA abundance. For example,
despite circadian transcription of LHCB1*3 (CAB1 ), a posttranscriptional mechanism of mRNA stabilization obscures
a rhythm in mRNA abundance (Millar and Kay 1991). In
contrast, the lack of circadian transcription of the Arabidopsis NIA2 nitrate reductase gene, as measured by nuclear
run-on experiments, suggests that the observed rhythm in
mRNA abundance reflects post-transcriptional control (Pilgrim et al. 1993). The chloroplast-encoded tufA gene of
Chlamydomonas exhibits a pronounced circadian oscillation
in mRNA abundance (Hwang et al. 1996). There is a
transcriptional component to this oscillation seen in lightdark (diurnal) cycles that persists into continuous light and
continuous dark. Additionally, there is an oscillation in the
stability of tufA mRNA in light-dark cycles. Although this
oscillation in post-transcriptional regulation does not persist
in continuous light and so is apparently not regulated by an
endogenous circadian oscillator, this represents a well-documented example of post-transcriptional regulation contributing to an oscillation in gene expression.
363
Rhythmic synthesis of both luciferase (Mittag et al. 1998)
and the luciferin binding protein (LBP) (Mittag et al. 1994)
of the dinoflagellate, Gonyaulax polyedra, results from circadian regulation of translation of mRNAs that do not oscillate in abundance (Morse et al. 1989, Mittag et al. 1998).
Interaction of an RNA-binding protein with the 3% untranslated region (UTR) is correlated with the translation of LBP
mRNA (Mittag et al. 1994). Excitingly, a protein capable of
binding to this 3% UTR element is conserved in Chlamydomonas, as is the circadian oscillation in abundance of this
binding activity (Mittag 1996). This should greatly facilitate
the genetic analysis of this post-transcriptional mode of
circadian regulation.
In Arabidopsis, overexpression of GRP7 blocks accumulation of the endogenous GRP7 and GRP8 transcripts, but
does not affect the rhythmic expression of several other
genes (Heintzen et al. 1997). This negative effect of GRP7,
an RNA-binding protein, on its own transcript abundance is
presumably through a mechanism of transcript destabilization because circadian transcription of the GRP7 promoter
is unaffected by overexpression of an ectopic copy of GRP7
(Staiger and Apel 1999).
Hormone production and responsiveness
The study of plant growth regulators has been complicated
for a number of reasons, and now several studies have
indicated that the additional complication of circadian regulation can no longer be ignored. Ethylene production shows
a circadian rhythm in barley, wheat and rye (Ievinsh and
Kreicbergs 1992), Chenopodium rubrum (Machácková et al.
1997) and sorghum (Morgan et al. 1997, Finlayson et al.
1998). In sorghum, it has been shown that this rhythm in
ethylene production reflects underlying rhythms in mRNA
abundance for the ACO2 gene encoding 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and in ACC oxidase activity (Finlayson et al. 1999). It was earlier
mentioned that there is a circadian oscillation in rosette leaf
auxin levels in Arabidopsis (Jouve et al. 1999). Through
heroic effort, a diurnal rhythm in gibberellin (GA) biosynthesis was demonstrated in sorghum (Foster and Morgan
1995); although this study did not address continuous conditions, it seems quite reasonable to suspect that there may be
circadian control over production of GA. Thus, circadian
control has been demonstrated over production of certainly
two and probably 3 of the major plant hormones, and
examples with other hormones may be forthcoming. It is
well established that there is circadian control over the
sensitivity of plants to light, for example, at the level of
stomatal aperture (Webb 1998) and in the induction of
LHCB and CAT gene expression (Millar and Kay 1996,
Zhong et al. 1998). It seems quite possible that plants will
exhibit circadian rhythmicity in their responsiveness to hormones. This might contribute to the rhythm in stomatal
aperture, even in the absence of a rhythm in abscisic acid
levels. Similarly, the rhythm in inflorescence stem elongation
in Arabidopsis in the absence of rhythmic IAA levels (Jouve
et al. 1999) might reflect rhythmic responsiveness to auxin.
364
Calcium
Free Ca2 + levels, monitored by aequorin luminescence in
transgenic tobacco and Arabidopsis, oscillate with a circadian rhythm in both the cytosolic and chloroplastic compartments (Johnson et al. 1995). This has significant
implications for all 3 aspects of circadian regulation, including both input and output pathways and the central oscillator itself. Ca2 + is a ubiquitous second messenger in plant
signaling pathways and has been implicated in phytochrome-mediated light signal transduction (Barnes et al.
1997) as well as in UV-B and UV-A/blue light-mediated
signaling (Christie and Jenkins 1996, Frohnmeyer et al.
1998, Long and Jenkins 1998). Pulses of blue light induce
transient spikes in cytosolic, but not in chloroplastic Ca2 + ,
and these transients are implicated in signaling downstream
of NON-PHOTOTROPIC HYPOCOTYL 1 (NPH1) in the
phototropic response (Baum et al. 1999). Ca2 + also plays a
critical role in guard cell signaling and so is implicated in the
circadian regulation of stomatal aperture and gas exchange
(McAinsh et al. 1997, Leckie et al. 1998).
Photoperiodism
The shift from vegetative to reproductive growth is a critical
and tightly regulated decision. Flowering time in many
species, including Arabidopsis, is environmentally-regulated
by photoperiod, light quality and vernalization (Koornneef
et al. 1998, Levy and Dean 1998, Lumsden and Millar 1998,
Piñeiro and Coupland 1998). Arabidopsis is a quantitative
long day plant because flowers are initiated more rapidly
under long than under short days. Mutations at many loci
affect the timing of flower initiation. These mutants can be
broadly grouped as early flowering or late flowering. Late
flowering mutants are more numerous, which may reflect the
preponderance of genetic studies in the common lab ecotypes Columbia (Col) and Landsberg erecta (Ler), both of
which are relatively early flowering. Genetic analyses suggest
3 different pathways promoting flowering: one constitutive,
one environmentally-responsive to photoperiod and to light
quality, and one responding to vernalization. Each pathway
negatively regulates a default state of floral repression.
In 1936, Bünning hypothesized that the circadian clock
was required for photoperiod sensitivity in flowering (Bünning 1936), and physiological and more recent genetic data
from Arabidopsis have corroborated his insight (Lumsden
and Millar 1998). Mutations that affect circadian rhythmicity frequently also affect flowering time. For example, timing
of CAB 1 (toc1 ) and elf 3 show defects in flowering time,
circadian gene expression and circadian leaf movement (Millar et al. 1995, Hicks et al. 1996, Somers et al. 1998b), and
early in short days 4 (esd4 ) shows defects in both flowering
time and leaf movement (Carré 1998). Both LHY and
CCA1 overexpressing mutants are late flowering (Schaffer et
al. 1998, Wang and Tobin 1998). Conversely, this suggests
that mutations affecting the photoperiodic pathway of flowering induction are candidates for also conferring circadian
defects. At least two examples of the utility of this approach
have been recently described. Mutations at the GIGANTEA
(GI) locus confer late flowering. GI has been recently cloned
Physiol. Plant. 109, 2000
(Fowler et al. 1999, Park et al. 1999) and GI transcript
abundance oscillates with a circadian rhythm that is altered
in elf 3, lhy and CCA1 overexpressing lines. Mutations in gi
affect rhythmicity of CCA1 and LHY transcripts (Fowler et
al. 1999, Park et al. 1999). A second example is seen with
null mutations of FLOWERING LOCUS C (FLC, also
known as FLF), a repressor of flowering that encodes a
MADS domain protein (Michaels and Amasino 1999, Sheldon et al. 1999), which confer early flowering and also
shorten the circadian period in leaf movement (Swarup et al.
1999). Collectively, these analyses suggest that there is an
intimate interaction between the circadian clock and photoperiodic timing and that the many mutations identified on
the basis of flowering time defects will also contribute
additional components of the circadian system.
Input to the clock
Light
By definition, circadian rhythms persist in the absence of
external time cues, but are entrainable to the environment.
The best-characterized input to the circadian clock is light.
Light pulses shift clock phase (Pittendrigh 1981a, Johnson
1992), although the resetting mechanisms vary among different organisms. For example, light resets the Neurospora
clock through an induction of FREQUENCY (FRQ) transcription (Crosthwaite et al. 1995, 1997), whereas light resets
the Drosophila clock through increased TIM protein degradation (Young 1998, Naidoo et al. 1999) and through a
sequestration of TIM by CRY (Ceriani et al. 1999).
A great deal is known about light perception in plants
(Chory 1997, Batschauer 1998, Casal et al. 1998, Whitelam
and Devlin 1998), although little is specifically known about
phototransduction to the clock, beyond the identity of key
photoreceptors. Light pulses phase shift clock-controlled
gene expression in wheat by a very low fluence phytochrome
response (Nagy et al. 1993). In addition to the acute effects
of pulses of light upon circadian phase, continuous applications of light of specific intensities and spectral qualities can
influence period length (Pittendrigh 1981a). A painstaking
analysis of the effects of either red or blue light of various
fluence rates on the period length of the circadian rhythm in
LHCB(CAB2 )::LUC expression has established that phytochrome B (PHYB) is the high-intensity red light photoreceptor for the clock whereas PHYA is the low-intensity red
light photoreceptor (Somers et al. 1998a). CRY1 is the
high-intensity blue light photoreceptor for the clock, and
both PHYA and CRY1 serve as low-intensity blue light
photoreceptors (Somers et al. 1998a). CRY2 (initially
defined genetically as the fha late flowering mutant) is
specifically implicated in the timing of flowering (Guo et al.
1998) and also contributes to the establishment of circadian
period (Somers et al. 1998a).
The understanding of the downstream signaling pathways
from PHY and CRY is far from complete (Deng and Quail
1999). Nucleoside diphosphate kinase as well as G-proteins
that function through cGMP or Ca2 + and Ca2 + -calmodulin pathways are implicated in red light signaling (Barnes et
Physiol. Plant. 109, 2000
al. 1997, Choi et al. 1999), and phosphorylation and Ca2 +
are implicated in blue light signaling (Jenkins 1997, Lasceve
et al. 1999). Recent studies have established a direct molecular interaction between PHYA and CRY1 in vitro (Ahmad
et al. 1998), offering a rationalization for co-action or
synergism between these signaling molecules (Mohr 1994).
Both PHYA and PHYB bind directly to PHYTCHROME
INTERACTING FACTOR 3 (PIF3), a PAS-domain putative bHLH transcription factor (Ni et al. 1998, Halliday et
al. 1999) and, critically, binding to PHYB is light-regulated
with the phytochrome signature of induction in response to
red light and reversibility by far red light (Ni et al. 1999). It
is intriguing that both phytochrome and PIF3 are PAS-domain proteins, given the prominence of PAS-domain
proteins in circadian systems of fungi, flies and mammals
(Dunlap 1999). PHYTOCHROME KINASE SUBSTRATE
1 (PKS1) is a substrate for light-regulated phosphorylation
by PHYA and PHYB (Fankhauser et al. 1999). Other
downstream signaling components are specific to PHYA or
to PHYB (reviewed by Deng and Quail 1999, von Arnim
1999). However, little is known of the signal transduction
cascade(s) leading specifically to the circadian clock (Kreps
and Kay 1997, Somers 1999). elf 3 was identified genetically
on the basis of a daylength-insensitive early flowering phenotype. elf 3 exhibits conditional arrhythmicity of both leaf
movement and LHCB gene expression in continuous light,
but shows normal clock function in continuous dark (Hicks
et al. 1996). Thus, ELF3 is interpreted as encoding a component of a light input pathway as opposed to a component of
a central oscillator.
Temperature
It is well established that temperature is as effective an
entraining stimulus as light (Bünning 1973), and recent data
have established that temperature is a stronger entraining
stimulus than light in Neurospora (Liu et al. 1998). Temperature provides a strong entraining input to rhythms in CO2
assimilation in CAM plants (Wilkins 1992). The mechanism
of temperature resetting in CAM plants is complex and
incompletely understood, but may involve alterations in
compartmentalization of malate (Grams et al. 1997). Cyclic
heat shocks can entrain mRNA oscillations in barley
seedlings (Kloppstech et al. 1991) and, more recently, temperature cycles have been shown to entrain LHCB transcription in Arabidopsis (Somers et al. 1998b). Cold pulses
reset the phase of the oscillations in LHCB and CCR2
(AtGRP7 ) mRNA (Kreps and Simon 1997). However, the
temperature step from 4 to 22°C, associated with release
from stratification, did not reset the circadian clock in
Arabidopsis, suggesting that very young seedlings are refractory to this temperature step (Zhong et al. 1998). In chilling
sensitive plants, such as tomato, it seems that cold pulses
actually stop the clock (Martino-Catt and Ort 1992, Jones et
al. 1998).
Other inputs to the central oscillator
A circadian oscillator is running in etiolated seedlings that
have not been exposed to light or temperature cycles or
365
pulses. For example, circadian oscillations have been detected in LHCB mRNA abundance in etiolated wheat
(Nagy et al. 1993) and tobacco (Kolar et al. 1995), and in
Arabidopsis LHCB1*1 (CAB2 ) transcription monitored either in etiolated transgenic tobacco seedlings (Millar et al.
1992a, Anderson et al. 1994) or in Arabidopsis (Millar and
Kay 1996). The amplitude of the acute induction of both
LHCB and CAT2 mRNA abundance by light varies strikingly according to the timing (phase) of the onset of illumination, which indicates that a circadian clock running in
etiolated seedlings regulates (gates) the induction of LHCB
and CAT2 by light (Millar and Kay 1996, Zhong et al.
1998). The acute induction of CAT2 mRNA varies with
time after imbibition, indicating that imbibition provides a
signal capable of resetting the circadian clock, although the
nature of this signal remains unknown (Zhong et al. 1998).
The oscillator in plants
Genetic and molecular approaches
The forward genetic approach (the hunt for mutants that
affect key clock properties, such as period length) has been
effective in the identification of clock components in other
systems (Dunlap 1999, Johnson and Golden 1999). In
plants, this genetic approach has emphasized Arabidopsis. A
genetic screen based on alterations in rhythmic expression of
a luciferase (LUC) transgene driven by regulatory elements
of the LHCB1*1 (CAB2 ) gene identified a series of timing of
CAB (toc) mutations that disrupt clock function (Millar et
al. 1995). The best-described of these mutants is toc1 -1,
which shortens the period of multiple rhythms, including
LHCB transcription (Millar et al. 1995), GRP7 (CCR2 )
mRNA accumulation (Kreps and Simon 1997), leaf movement, and stomatal conductance (Somers et al. 1998b). In
addition, toc1 -1 affects the photoperiodic flowering response, although this effect depends strongly on ecotype.
The most consistent aspect of the toc1 -1 flowering phenotype is a reduction of the difference between flowering times
in long versus short days (Somers et al. 1998b). Additional
toc1 alleles as well as other non-allelic toc loci have been
identified, but none of the genes identified by these mutations has yet been cloned. To date, TOC1 protein is probably the strongest known candidate for a component of the
central circadian oscillator, although the rigorous testing of
this hypothesis will require molecular manipulation that can
only be attempted once the TOC1 gene is cloned. A QTL
strategy, based on segregation of natural variation in the
circadian period length of leaf movement, has identified a
number of oscillator candidates in Arabidopsis, including
NON TROPPO, ANDANTE (possibly FLC), and
ESPRESSO and RALENTANDO, one of which may be GI
(Swarup et al. 1999).
late elongated hypocotyl (lhy) was identified in a screen for
late flowering mutations among an Arabidopsis population
carrying the maize Ac/Ds transposon system and was cloned
by virtue of the molecular Ds tag (Schaffer et al. 1998). The
Ds-tagged lhy mutants are functionally LHY overexpressors
in which the Ds element has inserted upstream of LHY,
366
resulting in constitutive high level expression (Schaffer et al.
1998). The most prominent feature of the LHY deduced
amino acid sequence is a single 47 residue region related to
the helix-turn-helix DNA-binding domain of the Myb family (Schaffer et al. 1998). This region is 87% identical to the
corresponding region of CCA1, which was identified by
virtue of its ability to bind to the DNA element of the
LHCB1*1 promoter shown to be sufficient to confer robust
circadian oscillations on reporter genes (Carré and Kay
1995, Wang et al. 1997). The similarity between LHY and
CCA1 extends beyond the Myb domain; they share 3 other
regions, each of 20 – 25 amino acids, that have at least 80%
identity (Schaffer et al. 1998, Wang and Tobin 1998). CCA1
and LHY are the founding members of a sub-family of
plant Myb proteins that have only single copies of the Myb
motif, in contrast to the vast majority of the numerous plant
Myb proteins [Arabidopsis has more than 80 (Romero et al.
1998)] in which the Myb domain is repeated.
The phenotype of plants carrying the dominant lhy allele
is pleiotropic and similar to the phenotype of plants overexpressing CCA1 (CCA1-ox) (Schaffer et al. 1998, Wang and
Tobin 1998). Overexpression of either LHY or CCA1 delays
flowering, consistent with disruption of the rhythmic expression of the late flowering gene, GI (Fowler et al. 1999), and
results in elongated hypocotyls. CCA1 and LHY mRNA
abundance oscillates with a circadian rhythm in wild type
plants, but becomes arrhythmic in overexpressing mutants.
Constitutive overexpression of either CCA1 or LHY results
in arrhythmicity of multiple clock outputs, including mRNA
abundance of all clock-regulated genes tested to date and
leaf movement (Schaffer et al. 1998, Wang and Tobin 1998).
These results, together with the sequence similarity between
LHY and CCA1, suggest that they might redundantly
specify central oscillator functions. However, loss of CCA1
function in a T-DNA disruptant line shortens the period of
mRNA oscillation in at least 3 clock-controlled genes
(LHCB, LHY and CAT2 ) and indicates that CCA1 and
LHY cannot be fully redundant (Green and Tobin 1999).
Moreover, CCA1 function cannot be required for oscillator
function, because the cca1 null plants are rhythmic (Green
and Tobin 1999).
Are either CCA1 or LHY components of a central oscillator? A set of criteria to define components of the circadian
oscillator have been proposed (Aronson et al. 1994) and
provide a helpful, if imperfect set of guidelines (see Foster
and Lucas 1999): (1) the activity (possibly, but not necessarily, the simple abundance) of a component should oscillate
with appropriate periodicity in the absence of external time
cues; (2) blocking the oscillation in the activity of a clock
component should abolish normal rhythmicity; (3) mutations in a clock component should affect canonical clock
properties, such as period length or temperature compensation, and null mutations should eliminate normal rhythmicity; (4) induced changes in a component’s activity should, by
feedback, change the component’s activity; (5) the overt
rhythm must be reset by induced changes in the activity of
a clock component; (6) the phase of component’s oscillation
should be reset by shifts in the light-dark regimen that reset
the clock.
For example, the mRNA and protein abundances of
GRP7 oscillate, satisfying criterion 1 (Heintzen et al. 1997).
Physiol. Plant. 109, 2000
However, overexpression of GRP7 in transgenic Arabidopsis
blocks the mRNA oscillation of GRP7 and GRP8, but does
not affect other circadian oscillations, satisfying criterion 5,
but failing criterion 2. Thus, GRP7 is interpreted to be a key
component of a slave (non-self-sustaining) oscillator, but
not of a central oscillator (Heintzen et al. 1997).
What then of CCA1 and LHY? CCA1 clearly plays a role
as an output component regulating circadian rhythmicity as
well as phytochrome responsiveness of LHCB transcription
(Wang et al. 1997). If CCA1 were an oscillator component,
then the output pathway from the oscillator to the LHCB
hand of the clock would be exceedingly short! It is also
possible that CCA1 is playing roles on both output and
input pathways (McClung 1998). If one systematically rates
CCA1 and LHY by the criteria outlined above, both oscillate at mRNA and protein levels and so both satisfy criterion 1. Overexpression studies indicate that both CCA1 and
LHY satisfy criterion 2. In each case, overexpression feeds
back to repress expression of the endogenous gene, which
satisfies criterion 4. The cca1 null mutation retains rhythmicity and, thus, CCA1 fails criterion 3. However, criterion
1 assumes that oscillator functions cannot be redundantly
specified, which seems unnecessarily restrictive. The remaining criteria have not been tested. The use of inducible
transgenes to provide pulses of CCA1 and LHY should
address criterion 5. Criterion 6 should also be testable
through phase shifting experiments.
However, it is important to recall that these criteria are
imperfect (Foster and Lucas 1999). Although the Neurospora FRQ locus satisfies these criteria (Aronson et al.
1994), recent experiments have established the presence of a
temperature entrainable circadian oscillator persisting in frq
null mutants and it is possible to reinterpret the FRQ
oscillator as participating in an input pathway (Roenneberg
and Merrow 1998, Merrow et al. 1999). Nonetheless, it
seems highly unlikely that anyone would argue that FRQ is
unimportant to the Neurospora circadian system. It may be
that, at least in the short term, one may have to be content
with the determination that any particular component is
important in the circadian system of an organism and defer
a decision on residence in input, oscillator or output compartments until one has a fuller understanding of the system
in question. Clearly CCA1 and LHY (as well as TOC1 and
ELF3 ) play important roles in the circadian system of
Arabidopsis.
As discussed earlier, the flowering time genes GI and FLC
also contribute to circadian timing. flc null mutations
shorten the period length in leaf movement (Swarup et al.
1999), but at this time there has been insufficient experimental manipulation of the expression of FLC to allow one to
draw conclusions about potential roles of FLC in the circadian system. gi mutants are altered in leaf movement and
gene expression rhythms of GI itself, and of LHCB, LHY
and CCA1 (Fowler et al. 1999, Park et al. 1999). In gi-2, a
null allele, the period of leaf movement is shortened, but the
period of gene expression rhythms gradually lengthens (Park
et al. 1999). Thus, it seems unlikely that GI represents an
oscillator component. The period shortening effect of gi-1
on gene expression rhythms is less severe in extended dark
than in continuous light. In addition, the extension of period
Physiol. Plant. 109, 2000
length seen in light of decreasing fluence is less pronounced
in gi-1 than in wild type (Park et al. 1999). Collectively,
these data suggest that GI acts on a light input pathway.
However, GI mRNA oscillates with a circadian rhythm,
making GI a clock output. Thus, one might best interpret
GI as part of an outer feedback loop necessary to sustain
both amplitude and period length of a central oscillator
(Park et al. 1999).
Phosphorylation
Differential phosphorylation of key clock components
(PER, TIM and FRQ) occurs throughout the circadian
cycle in Drosophila and Neurospora (Young 1998, Dunlap
1999). In Drosophila, DBT encodes a casein kinase Io activity responsible for the PER phosphorylation, and dbt null
mutants are arrhythmic (Kloss et al. 1998, Price et al. 1998).
Is there a role for phosphorylation in the activity of CCA1
or LHY? A yeast two-hybrid screen for Arabidopsis proteins
that interact with CCA1 identified a regulatory b subunit of
casein kinase II, CKB3 (Sugano et al. 1998). In vitro
experiments showed that CCA1 interacts with two other
casein kinase b subunits, CKB1 and CKB2, as well as with
two casein kinase catalytic (a) subunits, CKA1 and CKA2.
CK, as well as a CK2-like activity from Arabidopsis whole
cell extracts, phosphorylates CCA1 in vitro, although this
phosphorylation does not affect the ability of recombinant
CCA1 to bind to its DNA target in electrophoretic mobility
shift assays. Treatment of plant extracts with l protein
phosphatase abolished the formation of the major CCA1DNA complex, as did treatment of the extracts with CK2
inhibitors (Sugano et al. 1998). Overexpression of the CKB3
regulatory subunit in transgenic Arabidopsis increases CK2
activity, resulting in early flowering and shortening of the
period of the oscillations in mRNA abundance of CCA1
and LHY, as well as of 4 clock-controlled genes (LHCB1*1,
CCR2, CAT2 and CAT3 ) (Sugano et al. 1999). Clearly the
serine-threonine kinase activity of CK2 affects clock function, presumably via regulation of CCA1 activity, although
there may be other key targets (Sugano et al. 1999).
More than one clock?
A single cell, Gonyaulax polyedra, can house two distinct
circadian oscillators (Roenneberg and Morse 1993, Morse et
al. 1994). Have multiple oscillators been demonstrated in
multicellular plants? The rhythms in CO2 assimilation and
stomatal aperture exhibit a different period from the rhythm
in leaf movement in Phaseolus 6ulgaris, which indicates that
they are being driven by different oscillators, although this
may represent a single oscillator mechanism exhibiting a
difference in period between different organs (Hennessey
and Field 1992). The toc1 -1 mutation in Arabidopsis shortens the periods in LHCB transcription and leaf movement
to different degrees (Millar et al. 1995). Similarly, the gi-2
mutation shortens the period in leaf movement, but lengthens the period in gene expression (Park et al. 1999). In
neither example have the two rhythms been simultaneously
measured in the same plant, but the data suggest that
distinct oscillators, presumably in the distinct tissues, are
driving the two rhythms (Millar 1998). In extended dark367
ness, the period of LHCB transcription lengthens to about
30 h whereas the oscillations in mRNA abundance of CAT3
(Zhong et al. 1997), CCR1 (GRP8 ) and CCR2 (GRP7 )
(Carpenter et al. 1994) retain 24 h periods. Here the genes
are expressed in a single organ, the leaf, but it remains to be
established that the genes are being expressed within the
same cell. It has been recently shown that, in tobacco
seedlings in continuous red light, rhythms in cytosolic Ca2 +
and LHCB transcription exhibit different periods (Sai and
Johnson 1999). Again, it is difficult to establish that these
two rhythms are expressed in the same cells, but it is
nonetheless clear that the rhythms are responding to distinct
circadian oscillators (Sai and Johnson 1999). It is also
apparent that the rhythm in Ca2 + , as measured by Sai and
Johnson, cannot be responsible for the rhythmic transcription of LHCB (Sai and Johnson 1999), even though Ca2 +
has been implicated in phytochrome-mediated transcriptional control (Barnes et al. 1997).
Conclusions and perspectives
It is apparent that the relatively simple model of the circadian system presented in Fig. 1 is inadequate. Input pathways may change in sensitivity over the circadian cycle.
Outputs can feedback to provide input to the clock. Components can play multiple roles on input and output pathways,
and perhaps in the central oscillator itself. Moreover, we do
not yet have reliable criteria with which to unambiguously
assign molecules to roles as input, output or oscillator
components (Roenneberg and Merrow 1998, Merrow et al.
1999). Indeed, even the concept of a single central oscillator
has been revealed as inaccurate, as it is certain that a single
cell can contain two self-sustaining circadian oscillators
(Roenneberg and Morse 1993) as well as non-self-sustaining
slave oscillators (Heintzen et al. 1997). Whether there are
commonly multiple oscillators in a single cell or whether a
single molecular oscillator will behave differently in distinct
cell populations, tissues or organs remains to be determined
in most organisms. Nonetheless, it is evident that circadian
systems are complicated. Although this will be disappointing
to those who had hoped that the cloning of one or two key
‘clock genes’ would solve the puzzle, the rest of us are left
with the exhilarating challenge of unraveling a reticulate
network of output feedback loops providing input to interlocked oscillators.
Acknowledgements – I apologize to those whose work could not be
cited due to space limitations. I thank Todd Michael, Janet Painter,
Patrice Salomé, Lorenzo Sempere, Tom Jack and Mary Lou
Guerinot for comments on the manuscript. Work in my lab on
circadian rhythms is supported by grants from the National Science
Foundation (MCB 9723482 and IBN-9817603).
References
Ahmad M, Jarillo JA, Smirnova O, Cashmore AR (1998) The
CRY1 blue light photoreceptor of Arabidopsis interacts with
phytochrome A in vitro. Mol Cell 1: 939–948
Anderson SL, Kay SA (1995) Functional dissection of circadian
clock- and phytochrome-regulated transcription of the Arabidopsis CAB2 gene. Proc Natl Acad Sci USA 92: 1500 – 1504
368
Anderson SL, Teakle GR, Martino-Catt SJ, Kay SA (1994) Circadian clock- and phytochrome-regulated transcription is conferred by a 78 bp cis-acting domain of the Arabidopsis CAB2
promoter. Plant J 6: 457 – 470
Aronson BD, Johnson KA, Loros JJ, Dunlap JC (1994) Negative
feedback defining a circadian clock: autoregulation of the clock
gene frequency. Science 263: 1578 – 1584
Bae K, Lee C, Sidote D, Chuang K-Y, Edery I (1998) Circadian
regulation of a Drosophila homolog of the mammalian Clock
gene: PER and TIM function as positive regulators. Mol Cell
Biol 18: 6142 – 6151
Barnes SA, McGrath RB, Chua N-H (1997) Light signal transduction in plants. Trends Cell Biol 7: 21 – 26
Batschauer A (1998) Photoreceptors of higher plants. Planta 206:
479 – 492
Baum G, Long JC, Jenkins GI, Trewavas AJ (1999) Stimulation of
the blue light phototropic receptor NPH1 causes a transient
increase in cytosolic Ca2 + . Proc Natl Acad Sci USA 96: 13554–
13559
Bünning E (1936) Die endogene Tagesrhythmik als Grundlage der
photoperiodischen Reaktion. Ber Dtsch Bot Ges 54: 590–607
Bünning E (1973) The Physiological Clock, 3rd Ed. Springer, New
York, NY
Carpenter CD, Kreps JA, Simon AE (1994) Genes encoding
glycine-rich Arabidopsis thaliana proteins with RNA-binding
motifs are influenced by cold treatment and an endogenous
circadian rhythm. Plant Physiol 104: 1015 – 1025
Carré IA (1998) Genetic analysis of the photoperiod-sensing mechanism of the long-day plant Arabidopsis thaliana. In: Lumsden
PJ, Millar AJ (eds) Biological Rhythms and Photoperiodism in
Plants. BIOS Scientific Publishers, Oxford, pp 257–269
Carré IA, Kay SA (1995) Multiple DNA-protein complexes at a
circadian-regulated promoter element. Plant Cell 7: 2039–2051
Casal JJ, Sanchez RA, Botto JF (1998) Modes of action of phytochromes. J Exp Bot 49: 127 – 138
Ceriani MF, Darlington TK, Staknis D, Más P, Petti AA, Weitz
CJ, Kay SA (1999) Light-dependent sequestration of TIMELESS by CRYPTOCHROME. Science 285: 553 –556
Choi G, Yi H, Lee J, Kwon Y-K, Soh MS, Shin B, Luka Z, Hahn
T-R, Song P-S (1999) Phytochrome signalling is mediated
through nucleoside diphosphate kinase 2. Nature 401: 610–613
Chory J (1997) Light modulation of vegetative development. Plant
Cell 9: 1225 – 1234
Christie JM, Jenkins GI (1996) Distinct UV-B and UV-A/blue light
signal transduction pathways induce chalcone synthase gene
expression in Arabidopsis cells. Plant Cell 8: 1555–1567
Crosthwaite SK, Loros JJ, Dunlap JC (1995) Light-induced resetting of a circadian clock is mediated by a rapid increase in
frequency transcript. Cell 81: 1003 – 1012
Crosthwaite SK, Dunlap JC, Loros JJ (1997) Neurospora wc-1 and
wc-2 : transcription, photoresponses, and the origins of circadian
rhythmicity. Science 276: 763 – 769
Deng X-W, Quail PH (1999) Signalling in light-controlled development. Sem Cell Devel Biol 10: 121 – 129
Dowson-Day MJ, Millar AJ (1999) Circadian dysfunction causes
aberrant hypocotyl elongation patterns in Arabidopsis. Plant J
17: 63 – 71
Dunlap JC (1999) Molecular bases for circadian clocks. Cell 96:
271 – 290
Edmunds LN (1988) Cellular and Molecular Bases of Biological
Clocks. Springer-Verlag, New York, NY
Engelmann W, Johnsson A (1998) Rhythms in organ movement. In:
Lumsden PJ, Millar AJ (eds) Biological Rhythms and Photoperiodism in Plants. BIOS Scientific Publishers, Oxford, pp 35–50
Engelmann W, Simon K, Phen CY (1992) Leaf movement rhythm
in Arabidopsis thaliana. Z Naturforsch 47c: 925 – 928
Fankhauser C, Yeh K-C, Lagarias JC, Zhang H, Elich TD, Chory
J (1999) PKS1, a substrate phosphorylated by phytochrome that
modulates light signaling in Arabidopsis. Science 284: 1539–1541
Fejes E, Nagy F (1998) Molecular analysis of circadian clock-regulated gene expression in plants: features of the ‘output’ pathways. In: Lumsden PJ, Millar AJ (eds) Biological Rhythms and
Photoperiodism in Plants. BIOS Scientific Publishers, Oxford,
pp 99 – 118
Fejes E, Pay A, Kanevsky I, Szell M, Adam E, Kay S, Nagy F
(1990) A 268 bp upstream sequence mediates the circadian
clock-regulated transcription of the wheat Cab-1 gene in transgenic plants. Plant Mol Biol 15: 921 – 932
Physiol. Plant. 109, 2000
Feldman JF (1982) Genetic approaches to circadian clocks. Annu
Rev Plant Physiol 33: 583–608
Finlayson SA, Lee I-J, Morgan PW (1998) Phytochrome B and the
regulation of circadian ethylene production in sorghum. Plant
Physiol 116: 17–25
Finlayson SA, Lee I-J, Mullet JE, Morgan PW (1999) The mechanism of rhythmic ethylene production in sorghum. The role of
phytochrome B and simulated shading. Plant Physiol 119:
1083 – 1089
Foster KR, Morgan PW (1995) Genetic regulation of development
in Sorghum bicolor. IX. The maR
3 allele disrupts diurnal control
of gibberellin biosynthesis. Plant Physiol 108: 337 – 343
Foster RG, Lucas RJ (1999) Clocks, criteria and critical genes.
Nature Genetics 22: 217–219
Fowler S, Lee K, Onouchi H, Samach A, Richardson K, Morris B,
Coupland G, Putterill J (1999) GIGANTEA: a circadian clockcontrolled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several membrane-spanning
domains. EMBO J 18: 4679–4688
Frohnmeyer H, Bowler C, Zhu J-K, Yamagata H, Schäfer E, Chua
N-H (1998) Different roles for calcium and calmodulin in phytochrome and UV-regulated expression of chalcone synthase.
Plant J 13: 763–772
Giuliano G, Hoffman NE, Ko K, Scolnik PA, Cashmore AR (1988)
A light-entrained circadian clock controls transcription of several plant genes. EMBO J 7: 3635–3642
Glossop NRJ, Lyons LC, Hardin PE (1999) Interlocked feedback
loops within the Drosophila circadian oscillator. Science 286:
766 – 768
Gorton HL (1990) Stomates and pulvini: a comparison of two
rhythmic turgor-mediated movement systems. In: Satter RL,
Gorton HL, Vogelmann TC (eds) The Pulvinus: Motor Organ
for Leaf Movement. American Society of Plant Physiologists,
Rockville, MD, pp 214–237
Grams TEE, Borland AM, Roberts A, Griffiths H, Beck F, Lüttge
U (1997) On the mechanism of reinitiation of the endogenous
crassulacean acid metabolism rhythm by temperature changes.
Plant Physiol 113: 1309–1317
Green RM, Tobin EM (1999) Loss of the circadian clock-associated
protein 1 in Arabidopsis results in altered clock-regulated gene
expression. Proc Natl Acad Sci USA 96: 4176– 4179
Guo H, Yang H, Mockler TC, Lin C (1998) Regulation of flowering time by Arabidopsis photoreceptors. Science 279: 1360 – 1363
Halliday KJ, Hudson M, Ni M, Qin M, Quail PH (1999) poc1 : An
Arabidopsis mutant perturbed in phytochrome signaling because
of a T-DNA insertion in the promoter of PIF3, a gene encoding
a phytochrome-interacting bHLH protein. Proc Natl Acad Sci
USA 96: 5832–5837
Hao H, Glossop NRJ, Lyons L, Qiu J, Morrish B, Cheng Y,
Helfrich-Förster C, Hardin P (1999) The 69 bp circadian regulatory sequence (CRS) mediates per-like developmental, spatial,
and circadian expression and behavioral rescue in Drosophila. J
Neurosci 19: 987–994
Hartwell J, Smith LH, Wilkins MB, Jenkins GI, Nimmo HG (1996)
Higher plant phosphoenolpyruvate carboxylase kinase is regulated at the level of translatable mRNA in response to light or
a circadian rhythm. Plant J 10: 1071–1078
Heintzen C, Nater M, Apel K, Staiger D (1997) AtGRP7, a nuclear
RNA-binding protein as a component of a circadian-regulated
negative feedback loop in Arabidopsis thaliana. Proc Natl Acad
Sci USA 94: 8515–8520
Hennessey TL, Field CB (1991) Oscillations in carbon assimilation
and stomatal conductance under constant conditions. Plant
Physiol 96: 831–836
Hennessey TL, Field CB (1992) Evidence of multiple circadian
oscillators in bean plants. J Biol Rhythms 7: 105 – 113
Hicks KA, Millar AJ, Carré IA, Somers DE, Straume M, MeeksWagner DR, Kay SA (1996) Conditional circadian dysfunction
of the Arabidopsis early-flowering 3 mutant. Science 274: 790 –
792
Hwang S, Kawazoe R, and.Herrin DL (1996) Transcription of tufA
and other chloroplast-encoded genes is controlled by a circadian
clock in Chlamydomonas. Proc Natl Acad Sci USA 93: 996 –
1000
Ievinsh G, Kreicbergs O (1992) Endogenous rhythmicity of ethylene
production in growing intact cereal seedlings. Plant Physiol 100:
1389 – 1391
Physiol. Plant. 109, 2000
Jenkins GI (1997) UV and blue light signal transduction in Arabidopsis. Plant Cell Environ 20: 773 – 778
Johnson CH (1992) Phase response curves: what can they tell us
about circadian clocks? In: Hiroshige T, Honma K (eds) Circadian Clocks from Cell to Human. Hokkaido University Press,
Sapporo, pp 209 – 249
Johnson CH, Golden SS (1999) Circadian programs in cyanobacteria: adaptiveness and mechanism. Annu Rev Microbiol 53:
389 – 409
Johnson CH, Knight MR, Kondo T, Masson P, Sedbrook J, Haley
A, Trewavas A (1995) Circadian oscillations of cytosolic and
chloroplastic free calcium in plants. Science 269: 1863–1865
Johnson CH, Knight M, Trewavas A, Kondo T (1998) A clockwork
green: circadian programs in photosynthetic organisms. In:
Lumsden PJ, Millar AJ (eds) Biological Rhythms and Photoperiodism in Plants. BIOS Scientific Publishers, Oxford, pp 1–34
Jones TL, Ort DR (1997) Circadian regulation of sucrose phosphate
synthase activity in tomato by protein phosphatase activity.
Plant Physiol 113: 1167 – 1175
Jones TL, Tucker DE, Ort DR (1998) Chilling delays circadian
pattern of sucrose phosphate synthase and nitrate reductase
activity in tomato. Plant Physiol 118: 149 – 158
Jouve L, Greppin H, Agosti RD (1998) Arabidopsis thaliana floral
stem elongation: evidence for an endogenous circadian rhythm.
Plant Physiol Biochem 36: 469 – 472
Jouve L, Gaspar T, Kevers C, Greppin H, Agosti RD (1999)
Involvement of indole-3-acetic acid in the circadian growth of
the first internode of Arabidopsis. Planta 209: 136–142
Kehoe DM, Villand P, Somerville S (1999) DNA microarrays for
studies of higher plants and other photosynthetic organisms.
Trends Plant Sci 4: 38 – 41
Kellmann J-W, Merforth N, Wiese M, Pichersky E, Piechulla B
(1993) Concerted circadian oscillations in transcript levels of
nineteen Lha/b (cab) genes in Lycopersicon esculentum (tomato).
Mol Gen Genet 237: 439 – 448
Kellmann J-W, Hoffrogge R, Piechulla B (1999) Transcriptional
regulation of oscillating steady-state Lhc mRNA levels: characterization of two Lhca promoter fragments in transgenic tobacco
plants. Biol Rhythm Res 30: 264 – 271
Kim HY, Coté GG, Crain RC (1993) Potassium channels in
Samanea saman protoplasts controlled by phytochrome and the
biological clock. Science 260: 960 – 962
Kloppstech K (1985) Diurnal and circadian rhythmicity in the
expression of light-induced nuclear messenger RNAs. Planta
165: 502 – 506
Kloppstech K, Otto B, Sierralta W (1991) Cyclic temperature
treatments of dark-grown pea seedlings induce a rise in specific
transcript levels of light-regulated genes related to photomorphogenesis. Mol Gen Genet 225: 468 – 473
Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, Young
MW (1998) The Drosophila clock gene double-time encodes a
protein closely related to human casein kinase Ie. Cell 94:
97 – 107
Kolar C, Adám E, Schäfer E, Nagy F (1995) Expression of tobacco
genes for light-harvesting chlorophyll a/b binding proteins of
photosystem II is controlled by two circadian oscillators in a
developmentally regulated fashion. Proc Natl Acad Sci USA 92:
2174 – 2178
Kondo T, Ishiura M (1999) The circadian clocks of plants and
cyanobacteria. Trends Plant Sci 4: 171 – 176
Kondo T, Tsinoremas NF, Golden SS, Johnson CH, Kutsuna S,
Ishiura M (1994) Circadian clock mutants of cyanobacteria.
Science 266: 1233 – 1236
Koornneef M, Alonso-Blanco C, Peeters AJM, Soppe W (1998)
Genetic control of flowering time in Arabidopsis. Annu Rev
Plant Physiol Plant Mol Biol 49: 345 – 370
Kreps JA, Kay SA (1997) Coordination of plant metabolism and
development by the circadian clock. Plant Cell 9: 1235–1244
Kreps JA, Simon AE (1997) Environmental and genetic effects on
circadian clock-regulated gene-expression in Arabidopsis
thaliana. Plant Cell 9: 297 – 304
Lasceve G, Leymarie J, Olney MA, Liscum E, Christie JM,
Vavasseur A, Briggs WR (1999) Arabidopsis contains at least
four independent blue-light-activated signal transduction pathways. Plant Physiol 120: 605 – 614
Lecharny A, Wagner E (1984) Stem extension rate in light-grown
plants. Evidence for an endogenous circadian rhythm in
Chenopodium rubrum. Physiol Plant 60: 437 – 443
369
Lecharny A, Tremolières A, Wagner E (1990) Correlation between
the endogenous circadian rhythmicity in growth rate and fluctuations in oleic acid content in expanding stems of Chenopodium
rubrum L. Planta 182: 211–215
Leckie CP, McAinsh MR, Montgomery L, Priestley AJ, Staxen I,
Webb AAR, Hetherington AM (1998) Second messengers in
guard cells. J Exp Bot 49: 339–349
Levy YY, Dean C (1998) The transition to flowering. Plant Cell 10:
1973 – 1989
Liu Y, Merrow M, Loros JJ, Dunlap JC (1998) How temperature
changes reset a circadian oscillator. Science 281: 825 – 829
Liu Z, Taub CC, McClung CR (1996) Identification of an Arabidopsis Rubisco Activase (RCA) minimal promoter regulated by
phytochrome and the circadian clock. Plant Physiol 112: 43 – 51
Long JC, Jenkins GI (1998) Involvement of plasma membrane
redox activity and calcium homeostasis in the UV-B and UV-A/
blue light induction of gene expression in Arabidopsis. Plant Cell
10: 2077 – 2086
Lumsden PJ, Millar AJ (1998) Biological rhythms and photoperiodism in plants. Bios Scientific Publishers, Oxford
Machácková I, Chauvaux N, Dewitte W, van Onckelen H (1997)
Diurnal fluctuations in ethylene formation in Chenopodium rubrum. Plant Physiol 113: 981–985
Martino-Catt S, Ort DR (1992) Low temperature interrupts circadian regulation of transcriptional activity in chilling-sensitive
plants. Proc Natl Acad Sci USA 89: 3731–3735
Mayer W-E, Hohloch C, Kalkuhl A (1997) Extensor protoplasts of
the Phaseolus pulvinus: light-induced swelling may require extracellular Ca2 + influx, dark-induced shrinking inositol 1,4,5
triphosphate-induced Ca2 + mobilization. J Exp Bot 48: 219 –
228
McAinsh MR, Brownlee C, Hetherington AM (1997) Calcium ions
as second messengers in guard cell signal transduction. Physiol
Plant 100: 16–29
McClung CR (1997) The regulation of catalase in Arabidopsis. Free
Radic Biol Med 23: 489–496
McClung CR (1998) It’s about time: putative components of an
Arabidopsis circadian clock. Trends Plant Sci 3: 454 – 456
McClung CR, Hsu M, Painter JE, Gagne JM, Karlsberg, SD (2000)
Integrated temporal regulation of the photorespiratory pathway:
circadian regulation of two Arabidopsis genes encoding serine
hydroxymethyltransferase. Plant Physiol 123 (in press)
Merrow M, Brunner M, Roenneberg T (1999) Assignment of
circadian function for the Neurospora clock gene frequency.
Nature 399: 584–586
Michaels SD, Amasino RM (1999) FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of
flowering. Plant Cell 11: 949–956
Millar AJ (1998) The cellular organization of circadian rhythms in
plants: not one but many clocks. In: Lumsden PJ, Millar AJ
(eds) Biological Rhythms and Photoperiodism in Plants. BIOS
Scientific Publishers, Oxford, pp 51–68
Millar AJ, Kay SA (1991) Circadian control of cab gene transcription and mRNA accumulation in Arabidopsis. Plant Cell 3:
541 – 550
Millar AJ, Kay SA (1996) Integration of circadian and phototransduction pathways in the network controlling CAB gene transcription in Arabidopsis. Proc Natl Acad Sci USA 93:
15491 – 15496
Millar AJ, Short SR, Chua N-H, Kay SA (1992a) A novel circadian
phenotype based on firefly luciferase expression in transgenic
plants. Plant Cell 4: 1075–1087
Millar AJ, Short SR, Hiratsuka K, Chua N-H, Kay SA (1992b)
Firefly luciferase as a reporter of regulated gene expression in
higher plants. Plant Mol Biol Reporter 10: 324– 337
Millar AJ, Carré IA, Strayer CA, Chua N-H, Kay SA (1995)
Circadian clock mutants in Arabidopsis identified by luciferase
imaging. Science 267: 1161–1163
Mittag M (1996) Conseved circadian elements in phylogenetically
diverse algae. Proc Natl Acad Sci USA 93: 14401 – 14404
Mittag M, Lee D-H, Hastings JW (1994) Circadian expression of
the luciferin-binding protein correlates with the binding of a
protein to the 3% untranslated region of its mRNA. Proc Natl
Acad Sci USA 91: 5257–5261
Mittag M, Li L, Hastings JW (1998) The mRNA level of the
circadian regulated Gonyaulax luciferase remains constant over
the cycle. Chronobiol Int 15: 93–98
370
Mohr H (1994) Coaction between pigment systems. In: Kendrick
RE, Kronenberg GHM (eds) Photomorphogenesis in Plants, 2nd
Edition. Kluwer Academic Publishers, Dordrecht, pp 353–373
Morgan PW, Finlayson SA, Lee I-J, Childs KL, He C-J, Creelman
RA, Drew MC, Mullet JE (1997) Regulation of circadianly
rhythmic ethylene production by phytochrome B in sorghum. In:
Kanellis AK, Chang C, Kende H, Grierson D (eds) Biology and
Biotechnology of the Plant Hormone Ethylene. Kluwer Academic Publishers, Dodrecht, pp 105 – 111
Morse D, Hastings JW, Roenneberg T (1994) Different phase
responses of the two circadian oscillators in Gonyaulax. J Biol
Rhythms 9: 263 – 274
Morse D, Milos PM, Roux E, Hastings JW (1989) Circadian
regulation of bioluminescence in Gonyaulax involves translational control. Proc Natl Acad Sci USA 86: 172 –176
Nagy F, Kay SA, Chua N-H (1988) A circadian clock regulates
transcription of the wheat Cab-1 gene. Genes Dev 2: 376–382
Nagy F, Fejes E, Wehmeyer B, Dallman G, Schafer E (1993) The
circadian oscillator is regulated by a very low fluence response of
phytochrome in wheat. Proc Natl Acad Sci USA 90: 6290–6294
Naidoo N, Song W, Hunter-Ensor M, Sehgal A (1999) A role for
the proteasome in the light response of the Timeless clock
protein. Science 285: 1737 – 1741
Nakahira Y, Baba K, Yoneda A, Shiina T, Toyoshima Y (1998)
Circadian-regulated transcription of the psbD light-responsive
promoter in wheat chloroplasts. Plant Physiol 118: 1079–1088
Ni M, Tepperman JM, Quail PH (1998) PIF3, a phytochrome-interacting factor necessary for normal photo-induced signal transduction, is a novel basic helix-loop-helix protein. Cell 95:
657 – 667
Ni M, Tepperman JM, Quail PH (1999) Binding of phytochrome B
to its nuclear signalling partner PIF3 is reversibly induced by
light. Nature 400: 781 – 784
Nimmo HG (1998) Circadian regulation of a plant protein kinase.
Chronobiol Int 15: 109 – 118
Park DH, Somers DE, Kim YS, Choy YH, Lim HK, Soh MS, Kim
HJ, Kay SA, Nam HG (1999) Control of circadian rhythms and
photoperiodic flowering by the Arabidopsis GIGANTEA gene.
Science 285: 1579 – 1582
Piechulla B (1999) Circadian expression of the light-harvesting
complex protein genes in plants. Chronobiol Int 16: 115–128
Piechulla B, Merforth N, Rudolph B (1998) Identification of tomato
Lhc promoter regions necessary for circadian expression. Plant
Mol Biol 38: 655 – 662
Pilgrim ML, McClung CR (1993) Differential involvement of the
circadian clock in the expression of genes required for Ribulose1,5-bisphosphate carboxylase/oxygenase synthesis, assembly,
and activation in Arabidopsis thaliana. Plant Physiol 103: 553–
564
Pilgrim ML, Caspar T, Quail PH, McClung CR (1993) Circadian
and light regulated expression of nitrate reductase in Arabidopsis. Plant Mol Biol 23: 349 – 364
Piñeiro M, Coupland G (1998) The control of flowering time and
floral identity in Arabidopsis. Plant Physiol 117: 1–8
Pittendrigh CS (1981a) Circadian rhythms: entrainment. In: Aschoff
J (ed) Handbook of Behavioral Neurobiology. Biological
Rhythms. Plenum Press, New York, NY, pp 95 –124
Pittendrigh CS (1981b) Circadian rhythms: general perspective. In:
Aschoff J (ed) Handbook of Behavioral Neurobiology. Biological Rhythms. Plenum Press, New York, NY, pp 57–80
Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young MW
(1998) double-time is a novel Drosophila clock gene that regulates
PERIOD protein accumulation. Cell 94: 83 – 95
Roenneberg T, Merrow M (1998) Molecular circadian oscillators:
an alternative hypothesis. J Biol Rhythms 13: 167–179
Roenneberg T, Morse D (1993) Two circadian oscillators in one
cell. Nature 362: 362 – 364
Romero I, Fuertes A, Benito MJ, Malpica JM, Leyva A, Paz-Ares
J (1998) More than 80 R2R3 -MYB regulatory genes in the
genome of Arabidopsis thaliana. Plant J 14: 273 –284
Sai J, Johnson CH (1999) Different circadian oscillators control
Ca2 + fluxes and Lhcb gene expression. Proc Natl Acad Sci USA
96: 11659 – 11663
Salvador ML, Klein U, Bogorad L (1993) Light-regulated and
endogenous fluctuations of chloroplast transcript levels in
Chlamydomonas. Regulation by transcription and RNA degradation. Plant J 3: 213 – 219
Physiol. Plant. 109, 2000
Salvador ML, Klein U, Bogorad L (1998) Endogenous fluctuations
of DNA topology in the chloroplast of Chlamydomonas reinhardtii. Mol Cell Biol 18: 7235–7242
Satter RL, Gorton HL, Vogelmann TC (1990) The Pulvinus: Motor
Organ for Leaf Movement. American Society of Plant Physiologists, Rockville, MD
Scandalios JG, Guan L, Polidoros AN (1997) Catalases in plants:
gene structure, properties, regulation and expression. In: Scandalios JG (ed) Oxidative Stress and the Molecular Biology of
Antioxidant Defenses. Cold Spring Harbor Laboratory Press,
Plainville, NY, pp 343–406
Schaffer R, Ramsay N, Samach A, Corden S, Putterill J, Carré IA,
Coupland G (1998) LATE ELONGATED HYPOCOTYL, an
Arabidopsis gene encoding a MYB transcription factor, regulates circadian rhythmicity and photoperiodic responses. Cell 93:
1219 – 1229
Sheldon CC, Burn JE, Perez PP, Metzger J, Edwards JA, Peacock
WJ, Dennis ES (1999) The FLF MADS box gene: a repressor of
flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11: 445–458
Somers D (1999) The physiology and molecular bases of the plant
circadian clock. Plant Physiol 121: 9–19
Somers DE, Devlin P, Kay SA (1998a) Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock.
Science 282: 488–490
Somers DE, Webb AAR, Pearson M, Kay SA (1998b) The shortperiod mutant, toc1 -1, alters circadian clock regulation of multiple outputs throughout development in Arabidopsis thaliana.
Development 125: 485–494
Staiger D, Apel K (1999) Circadian clock-regulated expression of
an RNA-binding protein in Arabidopsis: characterisation of a
minimal promoter element. Mol Gen Genet 261: 811 – 819
Staiger D, Apel K, Trepp G (1999) The Atger3 promoter confers
circadian clock-regulated transcription with peak expression at
the beginning of night. Plant Mol Biol 40: 873– 882
Sugano S, Andronis C, Green RM, Wang Z-Y, Tobin EM (1998)
Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock-associated 1 protein. Proc Natl Acad Sci
USA 95: 11020–11025
Sugano S, Andronis C, Ong MS, Green RM, Tobin EM (1999) The
protein kinase CK2 is involved in regulation of circadian
rhythms in Arabidopsis. Proc Natl Acad Sci USA 96: 12362–
12366
Swarup K, Alonso-Blanco C, Lynn JR, Michaels SD, Amasino
RM, Koornneef M, Millar AJ (1999) Natural allelic variation
identifies new genes in the Arabidopsis circadian system. Plant J
20: 67 – 77
von Arnim AG (1999) Phytochrome in the limelight. Trends Plant
Sci 4: 465 – 466
Wang Z-Y, Tobin EM (1998) Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. Cell 93:
1207 – 1217
Wang Z-Y, Kenigsbuch D, Sun L, Harel E, Ong MS, Tobin EM
(1997) A Myb-related transcription factor is involved in the
phytochrome regulation of an Arabidopsis Lhcb gene. Plant Cell
9: 491 – 507
Webb AAR (1998) Stomatal rhythms. In: Lumsden PJ, Millar AJ
(eds) Biological Rhythms and Photoperiodism in Plants. BIOS
Scientific Publishers, Oxford, pp 69 – 79
Whitelam GC, Devlin PF (1998) Light signalling in Arabidopsis.
Plant Physiol Biochem 36: 125 – 133
Wilkins MB (1992) Circadian rhythms: their origin and control.
New Phytol 121: 347 – 375
Young MW (1998) The molecular control of circadian behavioral
rhythms and their entrainment in Drosophila. Annu Rev
Biochem 67: 135 – 152
Zhong HH, McClung CR (1996) The circadian clock gates expression of two Arabidopsis catalase genes to distinct and opposite
circadian phases. Mol Gen Genet 251: 196 – 203
Zhong HH, Resnick AS, Straume M, McClung CR (1997) Effects
of synergistic signaling by phytochrome A and cryptochrome 1
on circadian clock-regulated catalase expression. Plant Cell 9:
947 – 955
Zhong HH, Painter JE, Salomé PA, Straume M, McClung CR
(1998) Imbibition, but not release from stratification, sets the
circadian clock in Arabidopsis seedlings. Plant Cell 10: 2005–
2017
.
Edited by J. G. Scandalios
Physiol. Plant. 109, 2000
371